The TH 22-mediated IL-22 deficiency associated with premature ovarian insufficiency.

RESEARCH QUESTION: Is deficiency of IL-22 associated with premature ovarian insufficiency (POI)? DESIGN: Levels of IL-22 and IL-22BP, IL-22-producing T cells, and IL22RA1/IL10R2 expression were measured and compared among 29 patients with POI, 42 with precursor stage of POI (pre-POI) and 46 control women. Correlation of serum IL-22 and IL-22+ CD4+ T subsets with ovarian reserve markers were further analyzed. RESULTS: IL-22 levels in serum significantly differed among control women and patients with pre-POI and POI, with the lowest concentrations in POI group (p = .019). Significant reduction of peripheral CD4+ IL-22+ T cells was observed in patients with POI (p = .010), which mainly contributed by decrease of CD4+ IL-22+ IL-17- TH 22 cells (p = .012) but not TH 17 cells (p = .125). Levels of serum IL-22 and IL-22-producing CD4+ T subsets were significantly correlated with ovarian reserve markers, including AMH, bilateral AFC, follicle-stimulating hormone (FSH), and E2 (p < .05). The specific receptor IL22RA1 expression was marginally reduced in granulosa cells from patients with pre-POI (p = .051). No difference of IL-22BP was observed either in serum (p = .216) or follicular fluid (p = .856) among groups. CONCLUSIONS: Our study first demonstrated the significant association between TH 22-mediated IL-22 deficiency and ovarian insufficiency, which provide new insights into the autoimmune disturbance and opens new avenues for exogenous IL-22 administration as potential intervention of POI.

Autophagy regulates differentiation of ovarian granulosa cells through degradation of WT1.

Ovarian granulosa cells (GCs) proliferate and differentiate along with follicular growth, and this is indispensable for oocyte development and female fertility. Although the role of macroautophagy/autophagy in ovarian function has been reported, its contribution to the regulation of GC characteristics remains elusive. The siRNA-mediated knockdown of two key autophagy-related genes ATG5 and BECN1 and the autophagy inhibitor chloroquine were used to interfere with autophagy in GCs. Inhibition of autophagy both genetically and pharmacologically resulted in decreased expression of genes associated with GC differentiation, including CYP19A1/Aromatase and FSHR, as well as in reduced estradiol synthesis. Mechanistically, when autophagy was disrupted, the transcription factor WT1 accumulated in GCs due to its insufficient degradation by the autophagic pathway, and this inhibited GC differentiation. Finally, decreased expression of several autophagy-related genes, as well as reduced LC3-II:LC3-I and elevated SQSTM1/p62 protein levels, which are indications of decreased autophagy, were detected in GCs from biochemical premature ovarian insufficiency patients. In summary, our study reveals that autophagy regulates the differentiation of ovarian GCs by degrading WT1 and that insufficient autophagy might be involved in ovarian dysfunction.Abbreviations: ATG: autophagy related; bPOI: biochemical premature ovarian insufficiency; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CQ: chloroquine; E2: estradiol; FSH: follicle stimulating hormone; FSHR: follicle stimulating hormone receptor; GC: granulosa cell; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; POI: premature ovarian insufficiency; RAP: rapamycin; siRNA: small interfering RNA; WT1: WT1 transcription factor.

Treg deficiency-mediated TH 1 response causes human premature ovarian insufficiency through apoptosis and steroidogenesis dysfunction of granulosa cells.

Immune dysregulation has long been proposed as a component of premature ovarian insufficiency (POI), but the underlying mediators and mechanisms remain largely unknown. Here we showed that patients with POI had augmented T helper 1 (TH 1) responses and regulatory T (Treg ) cell deficiency in both the periphery and the ovary compared to the control women. The increased ratio of TH 1:Treg cells was strongly correlated with the severity of POI. In mouse models of POI, the increased infiltration of TH 1 cells in the ovary resulted in follicle atresia and ovarian insufficiency, which could be prevented and reversed by Treg cells. Importantly, interferon (IFN) -γ and tumor necrosis factor (TNF) -α cooperatively promoted the apoptosis of granulosa cells and suppressed their steroidogenesis by modulating CTGF and CYP19A1. We have thus revealed a previously unrecognized Treg cell deficiency-mediated TH 1 response in the pathogenesis of POI, which should have implications for therapeutic interventions in patients with POI.

New theca-cell marker insulin-like factor 3 is associated with premature ovarian insufficiency.

OBJECTIVE: To characterize circulating insulin-like factor 3 (INSL3) in different stages of ovarian insufficiency and its role in the evaluation of premature ovarian insufficiency (POI). DESIGN: Retrospective cohort study. SETTING: University-based center for reproductive medicine. PATIENT(S): A total of 145 women, including 48 patients with POI (25 IU/L < follicle-stimulating hormone [FSH] ≤40 IU/L), 49 with biochemical POI (bPOI) (10 IU/L < FSH ≤25 IU/L) and 48 age-matched control women with normal ovarian reserve (FSH <10 IU/L), retrospectively included from the reproductive hospital affiliated with Shandong University between 2017 and 2019. INTERVENTION(S): Levels of INSL3 in the serum and follicular fluid assayed with a commercial radioimmunoassay. MAIN OUTCOME MEASURE(S): Level of INSL3 in serum and follicular fluid among control women and patients with bPOI and POI, its association with different ovarian reserve markers, and its predictive value for bPOI and POI. RESULT(S): The serum INSL3 level continuously declined with the progress of ovarian insufficiency. It showed strong negative association with FSH (-0.655) and luteinizing hormone (-0.433), but positively correlated with antimüllerian hormone (0.617), inhibin B (0.400), antral follicle count (0.630), and testosterone (0.180). Additionally, the circulating INSL3 served as a good predictor for bPOI and POI. No statistically significant difference of INSL3 levels in follicular fluid was observed between bPOI patients and control women. CONCLUSION(S): For the first time our study has revealed an INSL3 deficiency in women with POI, indicating that circulating INSL3 could serve as a promising theca-cell specific marker for POI. Future research on the role of INSL3 in modulating follicular development, steroidogenesis, and POI pathogenesis is warranted.

Dysregulated cytokine profile associated with biochemical premature ovarian insufficiency.

PROBLEM: Premature ovarian insufficiency (POI) imposes great challenge on female reproduction. Whether immune disturbance in ovarian environment was implicated in POI remains unclear. We aimed to characterize the cytokine profile in follicular fluid (FF) and paired serum in patients with biochemical POI (bPOI). METHOD OF STUDY: Multiplex immunoassay containing 45 cytokines was performed for individual FF and paired serum samples from 35 bPOI patients and 37 matched controls. Cytokine profiles were compared between the two groups and cytokines correlated to ovarian reserve, and the rates of day-3 good-quality embryos were further analyzed. RESULTS: In FF, significantly elevated level of chemokines MIP-1α (P = .043), CXCL8 (P = .024), IP-10 (P = .041), and eotaxin-1 (P = .015) as well as growth factors VEGF-D (P = .047), BDNF (P = .043), LIF (P = .002), and bFGF (P = .046) was found in bPOI patients compared to controls. Yet RANTES manifested an opposite trend with reduced levels among bPOI patients (P = .006). All these chemokines and growth factors in FF were significantly correlated with ovarian reserve (P < .05). In paired serum, cytokine signature was not likely accordant with that in FF between two groups, except for increased IP-10 (P = .032) in bPOI patients and its significant correlation to FSH and AFC (P < .05). Among all differentially expressed cytokines, RANTES in FF was correlated with the rate of day-3 good-quality embryos (P = .035). CONCLUSION: Altered cytokine profile characterized by increased chemokines and growth factors was associated with early stage of POI, which may fuel the progression of the disease or even play a crucial role in the development of ovarian insufficiency.

Resumption of Ovarian Function After Ovarian Biopsy/Scratch in Patients With Premature Ovarian Insufficiency.

To evaluate the effect of ovarian biopsy and scratch on the resumption of ovarian function in women with premature ovarian insufficiency (POI), the follicle development and pregnancy outcome were retrospectively analyzed in women with POI after ovarian biopsy/scratch. Eighty patients with POI with secondary amenorrhea were accepted the laparoscopic surgery, and then hormone replacement treatment for 6 months was applied and in vitro fertilization and embryo transfer was suggested. No significant difference in clinical characteristics was observed before and after ovarian biopsy/scratch ( P > .05). After the ovarian surgery, 11 (13.75%) patients presented with ovarian function resumption spontaneously or with human menopausal gonadotropin stimulation. Three metaphases II oocytes were retrieved in 10 patients. Two embryos were formed and freshly transferred followed by a healthy singleton delivery in 1 (1.25%) patient. Patients with follicle development had higher baseline estradiol compared to those without (78.20 [18.53-161.08] pg/mL vs 15.70 [8.40-43.80] pg/mL, P < .01). The technique of ovarian biopsy/scratch could promote follicle development in vivo, suggesting it could bring promising benefits for some women with POI. However, future improvement in efficiency and practice criteria is warranted.

Impaired telomere length and telomerase activity in peripheral blood leukocytes and granulosa cells in patients with biochemical primary ovarian insufficiency.

STUDY QUESTION: Are telomere length and telomerase activity associated with biochemical primary ovarian insufficiency (POI)? SUMMARY ANSWER: Shortened telomere length and diminished telomerase activity were associated with biochemical POI. WHAT IS KNOWN ALREADY: POI is a result of pathological reproductive aging and encompasses occult, biochemical and overt stages. Studies have indicated telomere length as a biomarker for biological aging. STUDY DESIGN, SIZE, DURATION: A total of 120 patients with biochemical POI and 279 control women were recruited by the Center for Reproductive Medicine of Shandong University. PARTICIPANTS/MATERIALS, SETTING, METHODS: Telomere length in peripheral blood leukocytes (LTL) and granulosa cells (GTL) was measured using a modified Quantitative Polymerase Chain Reaction technique. The relative telomerase activity (RTA) in granulosa cells was detected using a modified quantitative-telomeric repeat amplification protocol assay. MAIN RESULTS AND THE ROLE OF CHANCE: After adjusting for age, patients with biochemical POI (n = 120) exhibited significantly shorter LTLs (0.75 ± 0.09 vs 1.79 ± 0.12, P < 0.001; OR = 0.54, 95% CI = 0.43-0.68) and GTLs (0.78 ± 0.09 vs 1.90 ± 0.23, P < 0.001; OR = 0.54, 95% CI = 0.41-0.70) than the controls (n = 279 for LTLs; n = 90 for GTLs). Significantly diminished RTAs in granulosa cells were detected in patients with biochemical POI (n = 31) compared with the controls (n = 38) (1.57 ± 0.59 vs 4.63 ± 0.93, P = 0.025; OR = 0.84, 95% CI = 0.72-0.98). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The cross-sectional nature of this study might have its limit in telomere length as well as telomerase activity along with the progressing decline in ovarian function. WIDER IMPLICATIONS OF THE FINDINGS: These findings suggest that telomere length and telomerase activity may be considered as indicators for progression of ovarian decline. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the National Basic Research Program of China (973 Program) (2012CB944700), Science research foundation item of no-earnings health vocation (201402004) and the National Natural Science Foundation of China (31471352, 81270662, 81471509, 81300461, 81522018). The authors have no potential conflict of interest to declare.

Novel mutations in the TP63 gene are potentially associated with Müllerian duct anomalies.

STUDY QUESTION: Are mutations and/or polymorphisms in the TP63 gene associated with human Müllerian duct anomalies (MDAs)? SUMMARY ANSWER: The novel mutation c.*374 G > A in the TP63 gene resulted in decreased expression of TP63 by generating new binding sites with miR-1260a/miR-532-3p and revealed the potential association between TP63 and human MDAs. WHAT IS KNOWN ALREADY: It has been shown that mice lacking Tp63 exhibit hypoplastic genitalia, a single cloacal opening, and persistence of columnar epithelium at lower genital tract sites. It has also been reported that a nonsense mutation in EMX2 results in decreased TP63 expression in a woman with MDAs. However, generally in humans the association between TP63 and MDAs is unknown. STUDY DESIGN, SIZE, DURATION: A total of 200 unrelated Chinese women with MDAs and 200 unrelated Chinese women with a normal uterus and vagina, as controls, were recruited in the Center for Reproductive Medicine of Shandong University. All participants had a normal karyotype (46, XX). PARTICIPANTS/MATERIALS, SETTING, METHODS: The 20 exons of the TP63 gene were sequenced in 200 cases and 200 controls. Putative binding sites for microRNAs were validated by dual luciferase activity assays. The role of microRNAs was further examined by western blot. MAIN RESULTS AND THE ROLE OF CHANCE: Sequence analysis revealed 15 known single-nucleotide polymorphisms. Additionally, three novel heterozygous variants, c.387 G > C, c.*374 G > A and c.*749 G > A, were identified in three patients with MDAs, none of which were detected in controls. Variant c.*374 G > A, located in the 3' untranslated region, was highly conserved among mammals and predicted to create microRNAs binding sites, which was confirmed by dual luciferase activity assays. Western blot demonstrated that the binding with miR-1260a/miR-532-3p resulting from the variation c.*374 G > A decreased the expression of TP63. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Further study is needed to uncover the role of the EMX2-TP63 pathway in the development of the Müllerian duct. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed the possible association between TP63 and MDAs and suggested a potential contribution of microRNA-regulated expression of genes in the etiology of MDAs. STUDY FUNDING/COMPETING INTERESTS: This research was supported by National Basic Research Program of China (973 Program) (2012CB944700), the State Key Program of National Natural Science Foundation of China (81430029), the National Natural Science Foundation of China (81270662, 81471509), China Postdoctoral Science Foundation (2014M561939) and the Scientific Research Foundation of Shandong Province of Outstanding Young Scientists (BS2014YY013, 2014BSE27022). The authors have no competing interests.

Antimüllerian hormone: correlation with age and androgenic and metabolic factors in women from birth to postmenopause.

OBJECTIVE: To study the age-specific distribution of antimüllerian hormone (AMH) and describe the association of AMH with androgenic and metabolic profiles at different ages. DESIGN: Cross-sectional study. SETTING: University hospital. PATIENT(S): A total of 6,763 Chinese women from birth to menopause. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Anthropometric parameters (height, weight, and blood pressure), and levels of AMH and testosterone, glucose metabolism, and lipid profiles. RESULT(S): According to the level of AMH, four age phases were established: childhood (0-10 years), adolescence (11-18 years), reproductive age (19-50 years), and advanced age (≥51 years). During childhood and adolescence, AMH levels increased, reaching a peak at 18 years. A decline occurred thereafter during the reproductive-age period until the age of 50 years, and it remained at a low level above 0 onward. We found that AMH was negatively correlated with testosterone in childhood (r = -0.25), but was positively correlated with testosterone and the free androgen index in adolescence (r = 0.30; r = 0.26, respectively) as well as during the reproductive phases (r = 0.28; r = 0.31, respectively). No correlation was observed between AMH and body mass index, fasting blood glucose, fasting insulin, the homeostasis model assessment, total cholesterol, triglycerides, low-density lipoprotein, or high-density lipoprotein at any phase. CONCLUSION(S): From birth to 18 years, AMH increases, then it declines thereafter, indicating changes of ovarian maintenance. A positive relationship between androgenic profiles and AMH during adolescence and reproductive years implies a synchronism between androgens and ovarian reserve.